Despite the high success rate, antiretroviral therapy does not cure the disease completely due to presence of latent viral reservoirs. Although several studies have addressed this issue earlier, the role of serum starvation/deprivation in HIV-1 latency has not been studied. So, we investigated the role of serum starvation in regulating HIV-1 latency. The impact of serum starvation on HIV-1 latency was assessed in latently infected monocytes U1 and T-cells J1.1. Serum starvation breaks HIV-1 latency in U1 cells. Under similar conditions, J1.1 cells failed to show reactivation of virus. We investigated the involvement of cell death pathway and autophagy during the serum starvation in viral reactivation. Inhibition of these pathways did not affect viral reactivation. Furthermore, other crucial factors like NF-κB, SP1 and AKT did not play any role in regulating viral latency. Here, we report that serum deprivation up-regulates ERK/JNK pathway. This leads to phosphorylation of c-Jun which plays an important role in viral reactivation. Treatment of cells with U0126, an ERK kinase inhibitor, potently inhibited viral replication. In summary, we show that serum starvation leads to reactivation of HIV-1 in latently infected monocytes through the ERK/JNK pathway.